European Journal of Pharmacology 562 (2007) 39 – 46

www.elsevier.com/locate/ejphar

Antinociceptive effect of intra-hippocampal CA1 and dentate gyrus injection

of MK801 and AP5 in the formalin test in adult male rats
Elaheh Soleimannejad a , Nasser Naghdi b , Saeed Semnanian a,c,⁎,
Yaghoub Fathollahi c , Anoshirvan Kazemnejad c
a
School of Cognitive Sciences, Institute for Studies in Theoretical Physics and Mathematics (IPM), Niavaran, P.O.Box 19395-5746,Tehran, Iran
b
Department of Physiology, Pasteur Institute of Iran, Tehran, Iran
c
Department of Physiology, Tarbiat Modares University, P.O. Box 14115-116, Tehran, Iran
Received 20 June 2006; received in revised form 10 November 2006; accepted 24 November 2006
Available online 1 December 2006

Abstract

Previous research has shown that the hippocampus processes pain related-information, probably through hippocampal neurons that respond
exclusively to painful stimulation. In the current experiments we tested whether blocking NMDA receptors in the hippocampal CA1 region and
dentate gyrus could reduce nociceptive behaviors in rats. The competitive and noncompetitive NMDA receptor antagonists 2-amino-5-
phosphonopentanoic acid (AP5; 3.75 μg/0.75 μl) and MK801 (1.5, 3, 6 μg/0.5 μl) were injected into the dentate gyrus and CA1 area of behaving
rats 5 min before subcutaneous injection of formalin irritant. Pain behaviors in both acute and tonic phases of the formalin test were significantly
reduced by AP5 (3.75 μg/0.75 μl) and MK801 (3 μg/0.5 μl, but not 1.5 and 6 μg/0.5 μl) injection to the dentate gyrus. In the CA1, injection of
AP5 had no effect while injection of the effective dose of MK801 (3 μg/0.5 μl) had a significant antinociceptive effect. This effect was apparent
only during the late phase of the formalin test. These results support the hypothesis that NMDA-sensitive mechanisms are involved in acute and
persistent pain-related processing in the dentate gyrus and with tonic pain processing in the hippocampal CA1 region.
© 2006 Elsevier B.V. All rights reserved.

Keywords: NMDA receptor; Formalin test; Hippocampus; Nociception; (Rat)

1. Introduction
Several physiological, pharmacological and behavioral lines of
evidence suggest that the hippocampal formation is involved in
nociception (Delgado, 1955; Khanna and Sinclaire, 1992;
McKenna and Melzack, 1992; Sinclair and Lo, 1986; Soleiman-
nejad et al., 2006). For example, the pyramidal cells and
interneurons in the dorsal hippocampal CA1 respond to persistent
noxious activation (Khanna, 1997; Khanna and Zheng, 1999);
Injection of local anaesthetic into the dentate gyrus of the
hippocampal formation produces an analgesic effect in the
formalin test (McKenna and Melzack, 1992); Hippocampal
lesions can cause avoidance task impairments in both animals
⁎ Corresponding author. Department of Physiology, Tarbiat Modares
University, P.O. Box 14115-116, Tehran, Iran. Tel.: +98 21 8801 1001 #
4520; fax: +98 21 8801 3030.
E-mail address: ssemnan@modares.ac.ir (S. Semnanian).
0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejphar.2006.11.051
(Nadel, 1968; Olton and Issacson, 1968) and humans; Partial
hippocampectomy has been used (with moderate success) as a
treatment for chronic pain (Gol and Faibish, 1967), whereas
electrical stimulation of the hippocampal formation evokes painful
sensations in humans (Delgado, 1955; Gloor et al., 1981; Halgren
et al., 1978); Blocking neural transmission along the major
afferent (McKenna and Melzack, 1992) or efferent (Vaccarino and
Melzack, 1992) hippocampal pathways has reduced pain
behaviors; Peripheral noxious stimulation alters the induction of
Fos (Aloisi et al., 1997; Funahashi et al., 1999) and Egr1 (Pearse et
al., 2001; Wei et al., 2000) in the hippocampal formation, Fos and
Egr1 being transcription proteins that are expressed in neurons
following synaptic excitation (Aloisi et al., 1997; Khanna et al.,
2004); Finally, the hippocampus is also assumed to play an
important role in the affective and motivational components of
pain perception (Henke, 1982; Melzack and Casey, 1968).
NMDA (N-methyl-D-aspartate) receptors are localized both
in supraspinal (hippocampus, cerebral cortex, thalamus,
40 E. Soleimannejad et al. / European Journal of Pharmacology 562 (2007) 39–46
striatum, cerebellum, and brain stems) and spinal (substantia
gelatinosa and spinal gray matter) structures (Kalb and Fox,
1997; Mugnaini et al., 1996; Roth et al., 1996). The NMDA
receptor plays a key role in central pain transduction
mechanisms (D'Amico et al., 1996; Hudespith, 1997). Numer-
ous studies have focused on NMDA receptor activation in pain-
related neuroplasticity, especially within the spinal cord
(Coderre and van Empel, 1994a,b; Dickenson et al., 1997).
The excitatory amino acids glutamate and aspartate are involved
in the processing of nociceptive information in the spinal cord.
In particular, they facilitate and enhance the excitability of
nociceptive inputs from spinal cord neurons to the central
nervous system during persistent pain (Hudespith, 1997;
Maione et al., 1999, 2000). NMDA receptor antagonists
attenuate pain behaviors in models of neuropathic (Seltzer
et al., 1991; Walters, 1987) and tonic (Coderre and Melzack,
1992a,b; Coderre and van Empel, 1994a,b; Eisenberg et al.,
1993; Klepstad et al., 1990) pain, when applied to the CNS.
Clinical studies have also shown that various NMDA receptor
antagonists are useful analgesics in the treatment of acute
(Henderson et al., 1999; Schmid et al., 1999) and neuropathic
(Enarson et al., 1999; Klepstad et al., 1990; Kristensen et al.,
1992; Pud et al., 1998; Rabben et al., 1999) pain syndromes.
Electrophysiological responses of nociceptive neurons in the
ventrobasal thalamus can be blocked by NMDA antagonists
(Eaton and Salt, 1987, 1990; Salt et al., 1988); likewise,
behavioral pain responses have been blocked by AP5 (2-amino-
5-phosphonovalerate) applied to the centromedial thalamus
(McKenna and Melzack, 1994). Finally, microinjection of the
NMDA receptor antagonist AP5 into the dentate gyrus region of
the hippocampus attenuated pain behaviors in both the acute
and tonic phases of the formalin test, but had no effect when
administered into the hippocampal CA1 region, the cortex or the
cerebellar ventricles (McKenna and Melzack, 2001).
The formalin model of inflammatory pain is probably better
than phasic mechanical or thermal stimuli tests in modeling
human pain (Abbott et al., 1995; Tjolsen et al., 1992). During
the late phase of the formalin test the spinal cord releases
excitatory amino acids and NMDA receptor subtypes are
activated (Coderre and van Empel, 1994a). Intrathecal injection
of selective NMDA antagonists prevents the nociceptive
behaviour of the late phase (Coderre and van Empel, 1994a;
Eisenberg et al., 1993).
In the current study we investigated the role of NMDA-
sensitive mechanisms at the hippocampal CA1 and dentate
gyrus, using in the formalin model of persistent pain. For this
purpose, intra-CA1 and intra-dentate gyrus injections of a
competitive (AP5) and noncompetitive (MK801) antagonist
NMDA were used.
2. Materials and methods
Experiments were approved by the Institute for Studies in
Theoretical Physics and Mathematics in Teheran and conducted
according to its regulations. Experiments adhered to the
guidelines for animal experimentation of the International
Association for the Study of Pain (Zimmermann, 1983).
2.1. Animals
Male albino rats of Wistar strain, weighing 250–280 g, were
used as subjects. Rats were housed four per cage in a
temperature and light-controlled room under a 12:12 h light:
dark cycle with water and food provided ad libitum.
2.2. Surgery
Rats (n = 7/group) were anesthetized with a mixture of
ketamine and xylazine (100 mg/kg i.p.). Two guide cannulae
(22-gauge) were implanted bilaterally into the CA1 region of
the hippocampus at the following stereotaxic coordinates:
− 3.72 mm posterior to bregma, ± 2.2 mm laterally and 2.4 mm
dorsal-ventrally, ventral to the outer skull surface, or into the
dentate gyrus region of the hippocampus at the following
coordinates: − 3.72 mm posterior to bregma, ± 2.2 mm laterally
and 3.4 mm dorsal-ventrally, ventral to outer skull surface.
Cannulae were held in place with dental acrylic applied around
them and two anchoring screws.
2.3. Microinjection procedure
Drugs and vehicle were administered into the CA1 region of
the hippocampus or into the dentate gyrus through guide
cannulae using injection needles (27-gauge) connected by
polyethylene tubing to 10 μl Hamilton microsyringe. The
injection needle was inserted 0.5 mm beyond the tip of the
cannula and vehicle (saline) or various doses of the tested
compounds (MK801 or AP5, Sigma co.) were injected over
3 min.
2.3.1. Experiment 1
The aim of this experiment was to investigate the role of
NMDA receptors in the CA1 region of hippocampus on
nociception. Rats with cannula aimed at the CA1 were divided
into four groups and treated with either saline (n = 7) or three
doses of MK801 (1.5, 3, 6 μg/0.5 μl; Tucci et al., 1998), injected
bilaterally into the CA1 region. Five minutes after intra-CA1
injection of 0.5 μl saline or MK801 the formalin test was
performed.
2.3.2. Experiment 2
The aim of this experiment was to investigate the role of
NMDA receptors in the CA1 region of hippocampus on
nociception. Rats with cannula aimed at the CA1 were divided
into two groups (n = 14). Saline (0.75 μl) or AP5 (3.75 μg/
0.75 μl; McKenna and Melzack, 2001) were injected bilaterally
into the CA1 region. Five minutes later the formalin test was
performed.
2.3.3. Experiment 3
The aim of this experiment was to investigate the role of
NMDA receptors in the dentate gyrus on nociception. Rats with
cannula aimed at the dentate gyrus were divided into four
groups and treated with either saline (n = 7) or three doses of
MK801 (1.5, 3, 6 μg/0.5 μl; Tucci et al., 1998), injected
 E. Soleimannejad et al. / European Journal of Pharmacology 562 (2007) 39–46
bilaterally into the dentate gyrus. Five minutes after intra-CA1
injection of 0.5 μl saline or MK801 the formalin test was
performed.
2.3.4. Experiment 4
The aim of this experiment was to investigate the role of
NMDA receptors in the dentate gyrus of hippocampus on
nociception. Rats with cannula aimed at the CA1 were divided
into two groups (n = 14). Saline (0.75 μl) or AP5 (3.75 μg/
0.75 μl; McKenna and Melzack, 2001) were injected bilaterally
into the dentate gyrus. Five minutes later the formalin test was
performed.
2.4. Formalin test
Seven days after surgery, each rat was placed in a transparent
acrylic cage and was allowed to move freely for 15–20 min to
Fig. 1. Schematic picture of the injection sites. The injection of AP5 into the CA1 (7 sites; ▲, ● and ■ signs) and dentate gyrus (7 sites;
− 3.72 and − 3.84 bregmas (from up to down) are shown.
41
habituate. A mirror was placed under the cage to allow an
unobstructed view of the animal's paws to the behavioral
observer. Five minutes after intra-hippocampal injections of
either vehicle, MK801 or AP5, each rat was restrained and
received a 50-μl subcutaneous injection of 5% buffered formalin
acetate into the left hind-paw and placed in the observation box
for 60 min. The pain response in the formalin test consists of an
initial display of nociceptive behaviors (described below) that
subsides after approximately 5 min and reappears after an
additional 10–15 min; it then slowly diminishes over the
subsequent 40–60 min. Subjects in this experiment were
observed for 60 min following formalin injection and each
15 s were continuously rated by a 4-point scale:
A score of 0 denotes normal use of the injected paw (i.e., the
plantar surface of the paw comes into full contact with the floor
of the observation box and the animal's weight is evenly
distributed between hind paws).
◆, ◣ and ▼ signs) in − 3.60,
42 E. Soleimannejad et al. / European Journal of Pharmacology 562 (2007) 39–46
Fig. 2. Formalin test: the effect of MK801 injected into the CA1 region. Data
are expressed as the scores of nociceptive behavior induced by formalin
injection (50 μl at 5%) in rats treated with MK801 (1.5, 3 and 6 μg/0.5 μl)
5 min before formalin administration. Each animal was observed for 60 min
after formalin injection. Each point is the mean ± SEM of the accumulative
time of nociceptive behavior/5 min. The comparison was done between
vehicle and MK801-treated groups (n = 7). The effective dose of MK801
(3 μg/0.5 μl) had a significant antinociceptive effect only during 10 and 40
timepoints. MK801 had no effect in other time periods or in different doses
(⁎p b 0.05 vs. vehicle).
A score of 1 indicates careful use of the injured paw, with
some part of the paw in contact with the floor; the animal limps
when walking.
A score of 2 indicates elevation of the paw.
A score of 3 denotes vigorous shaking or licking of the
injured paw (distinct from normal grooming behavior).
Scores of each timepoint (5 min intervals) were calculated by
the following equation:
0
T0 þ 1
T1 þ 2
T2 þ 3
T3
¼ Score
300
In this equation T0–T3 designates the number of times rats
are graded scores of 0–3, respectively. This way, pain behaviors
are expressed during every 5 min in the time course data during
Fig. 3. Average pain scores during the early (0–5 min) and late (25–60 min)
phases of the formalin test. Rats received bilateral injections of MK801 into the
CA1 region of hippocampus. The effective dose of MK801 (3 μg/0.5 μl) had a
significant anti-nociceptive effect in the tonic phase only (⁎p b 0.05 vs. vehicle).
Fig. 4. Formalin test: the effect of AP5 injected into the CA1 region. Data are
expressed as the scores of nociceptive behavior induced by formalin injection
(50 μl at 5%) in rats treated with AP5 (3.75 μg/0.75 μl) 5 min before formalin
administration. Each animal was observed for 60 min after formalin injection.
Each point is the mean ± SEM of the accumulative time of nociceptive behavior/
5 min. The comparison was done between vehicle and AP5-treated groups
(n = 7). AP5 injected into the CA1 region had no effect in the behavior scores of
the formalin test (⁎p b 0.05).
the initial acute phase (0–5 min) or the second, tonic phase (20–
60 min; Coderre et al., 1990; Cohen et al., 1984).
2.5. Histology
Approximately 1 h after being tested, animals were
administered an overdose of ether and their brains were
removed and stored in 10% formalin for 48 h. Cannulae
placements were verified by performing 100 μm coronal
sections. Each subject's data were included in the statistical
analysis only if cannula tips were just above the aimed region
and the injection tracks were exactly spread in a limited area in
the CA1 or dentate gyrus of the hippocampus (Fig. 1).
2.6. Statistical analysis
The effects of drugs were analyzed by one-way and repeated
measure ANOVA for the phase data (Figs. 3, 5, 7 and 9) and the
time course data (Figs. 2, 4, 6 and 8) respectively. Post hoc
analyses were made by LSD test. All results are presented as
Fig. 5. Average pain scores during the early (0–5 min) and late (25–60 min)
phases of the formalin test. Rats received bilateral injections of AP5 into the
CA1 region of hippocampus. The AP5 (3.75 μg/0.75 μl) had no effect in the
acute and tonic phases (⁎p b 0.05 vs. vehicle).
 E. Soleimannejad et al. / European Journal of Pharmacology 562 (2007) 39–46
Fig. 6. Formalin test: the effect of MK801 injected into the dentate gyrus. Data
are expressed as the scores of nociceptive behavior induced by formalin
injection (50 μl at 5%) in rats treated with MK801 (1.5, 3 and 6 μg/0.5 μl) 5 min
before formalin administration. Each animal was observed for 60 min after
formalin injection. Each point is the mean ± SEM of the accumulative time of
nociceptive behavior/5 min. The comparison was done between vehicle and
MK801-treated groups (n = 7). The effective dose of MK801 (3 μg/0.5 μl) had a
significant antinociceptive effect during 5, 20, 25, 30, 35, 40, 45, 50 and 60
timepoints after formalin injection (⁎p b 0.05 vs. vehicle).
means ± S.E.M. In all statistical comparisons, P values b 0.05
were considered to be significant.
3. Results
3.1. Experiment 1
Formalin injection into the left paw resulted in the typical
biphasic response (Fig. 2). The NMDA antagonist MK801
injected bilaterally into the CA1 hippocampal region 5 min
before the formalin test had a significant effect only at a dose of
3 μg/0.5 μl, compared to the saline control group. This effect
was apparent during the late phase only (25–60 min, F4,30 = 3.5;
p b 0.019; Fig. 3), and during the 10 and 40 timepoints (Fig. 2).
3.2. Experiment 2
Formalin injection into the left paw of rat resulted in the
typical biphasic response (Fig. 4). The NMDA antagonist
Fig. 7. Average pain scores during the early (0–5 min) and late (25–60 min)
phases of the formalin test. Rats received bilateral injections of MK801 into the
dentate gyrus The effective dose of MK801 (3 μg/0.5 μl) had a significant
antinociceptive effect in the acute and tonic phases (⁎p b 0.05 vs. vehicle).
43
Fig. 8. Formalin test: the effect of AP5 injected into the dentate gyrus. Data are
expressed as the scores of nociceptive behavior induced by formalin (50 μl at
5%) in rats treated with AP5 (3.75 μg/ 0.75 μl) 5 min before formalin
administration. Each animal was observed for 60 min after formalin injection.
Each point is the mean ± SEM of the accumulative time of nociceptive behavior/
5 min. The comparison was done between vehicle and AP5-treated groups
(n = 7). AP5 had a significant antinociceptive effect during 5, 25, 45, 55 and 60
timepoints (⁎p b 0.05 vs vehicle).
AP5 (3.75 μg/0.75 μl) injected bilaterally into the CA1
hippocampal region 5 min before the formalin test had no
effect during the early (0–5 min) and late (25–60 min) phases
of the formalin test, as compared to the saline control group
(Fig. 5).
3.3. Experiment 3
Formalin injection into the left paw resulted in a typical
biphasic response (Fig. 6). Compared to the saline group,
bilateral injection of 3 μg/0.5 μl MK801 into the dentate gyrus
5 min before the formalin test resulted in a significant decrease
in the overall pain behavior scores of rats. This effect of
MK801was apparent in both the acute (0–5 min, F4,26 = 2.9;
p b 0.04) and tonic (25–60 min, F4,26 = 3.5; p b 0.02) intervals
of the formalin test (Fig. 7) and during the 5, 20, 25, 30, 35, 40,
45, 50, 60 timepoints in the time course data (Fig. 6). In other
concentrations (1.5 and 6 μg/0.5 μl) MK801 had no effect on
pain behavior.
Fig. 9. Average pain scores during early (0–5 min) and late (25–60 min) phases
of the formalin test. Rats received bilateral injections of AP5 into the dentate
gyrus. AP5 had a significant antinociceptive effect in the acute and tonic phases
(⁎p b 0.05 vs. vehicle).
44 E. Soleimannejad et al. / European Journal of Pharmacology 562 (2007) 39–46
3.4. Experiment 4
Formalin injection into the left paw of the rat resulted in the
typical biphasic response (Fig. 8). Compared to the saline
group, bilateral injection of the NMDA antagonist AP5
(3.75 μg/0.75 μl) into the dentate gyrus 5 min before the
formalin test resulted in a significant decrease in the overall pain
behavior scores of rats. This effect was apparent in both the
acute (0–5 min, F2,16 = 4.6; p b 0.026) and tonic (25–60 min,
F2,16 = 4.3; p b 0.03) intervals of the formalin test (Fig. 9) and
during the 5, 25, 45, 55, 60 timepoints (Fig. 8).
4. Discussion
These results indicate that the competitive and noncompet-
itive NMDA antagonists AP5 and MK801 administered into the
dentate gyrus significantly reduce pain behavior in both the
acute and tonic phases of the formalin test. In contrast, injection
of these compounds into the CA1 region of the hippocampus
was significantly less effective: AP5 had no significant effect on
nociceptive behaviors both in the acute and tonic phases,
whereas only one of the three tested doses of MK801 (3 μg)
changed nociceptive behavior of rats in the tonic, but not the
acute phase of the formalin test.
Previous studies, including a study performed in our lab,
have shown that the hippocampal formation is involved in
nociception (Delgado, 1955; Khanna and Sinclaire, 1992;
McKenna and Melzack, 1992; Seltzer et al., 1991; Soleiman-
nejad et al., 2006). Granular cells in the dentate gyrus are
glutamatergic and GABAergic and receive synapses from
neurons of the entorhinal cortex through the perforant pathway
(Amaral and Witter, 1995; Vizi and Kiss, 1998). In turn, these
granular cells send their axons into the CA3 area of the
hippocampus via the mossy fiber pathway (Amaral and Witter,
1995). Granular cells axon terminals in the CA3 area synapse
onto both glutamatergic pyramidal neurons and GABAergic
interneurons (Amaral and Witter, 1995; Vizi and Kiss, 1998). In
strata radiatum and oriens of the hippocampal CA1 area, most
glutamate-releasing nerve terminals derive from axon collat-
erals of CA3 pyramidal cells. The two main targets of this input
are the spines of pyramidal cells and the dendritic shafts of the
different kinds of interneurons. Fast synaptic transmission in
these synapses is mostly mediated by AMPA- and NMDA-type
glutamate receptors (Baude et al., 1995; Nusser et al., 1998a;
Racca et al., 2000).
In dentate gyrus NMDA antagonists may act on glutamater-
gic granular cells and inhibit the activity of these cells. This
support previous finding that disruption of neural activity in the
dentate gyrus reduces formalin pain (McKenna and Melzack,
1992).
Our results show that the NMDA antagonist MK801,
injected into the CA1area of the hippocampus, reduces pain
during the late phase after formalin injection. Since the CA1
GABAergic interneurons have NMDA receptors (see above;
Baude et al., 1995; Nusser et al., 1998a; Racca et al., 2000) it is
possible that MK801 exerts its antinociceptive effect by
inhibiting these interneurons in the hippocampal CA1 area.
This suggestion support previous findings that subcutaneous
formalin increased firing rate of a majority of the field CA1
putative GABAergic interneurons and induced a long lasting
depression of synaptic excitability of CA1 pyramidal cell
(Khanna, 1997; Khanna and Zheng, 1999).
The present data show that AP5, injected into the CA1 area
of hippocampus, had no significant effect on nociceptive
behaviors both in the acute and tonic phases. Our findings are in
agreement with McKenna's finding that AP5 injections at sites
in the CA region did not reduce overall pain behavior
(McKenna and Melzack, 2001). In contrast to the AP5 data,
we found that MK801 reduces nociception in the second phase
of the formalin test when injected into the CA1 area, supporting
the anti-nociceptive role of NMDA antagonist when applied
into other brain sites (McKenna and Melzack, 2001). It is
possible, therefore, that MK801, being a noncompetitive
antagonist of NMDA receptors, exert significant anti-nocicep-
tive effect in a dose of 3 μg but not 1.5 or 6 μg, in contrast to the
AP5 effect. This difference between MK801 and AP5 is
supported by previous study, showing that they differed in their
ability to protect against hippocampal hypoxic neuronal damage
(Schurr et al., 1995). This difference could be explained by
differential activity at the receptor site (Hardman et al., 1996).
The present study confirms that the CA1 region of
hippocampus and dentate gyrus is involved in neural processing
related to persistent pain. AP5 or MK801 injected into the
dentate gyrus may attenuate directly or indirectly acute and
tonic nociceptive signals to forebrain, or other brain structures,
receiving efferent outflow from the hippocampal formation. It
seems that NMDA receptors in the CA1 region may have a role
in persistent nociceptive behavior. The precise mechanism(s)
through which MK801 causes reduced nociceptive behavior is
not clearly understood at present.
In conclusion, our data suggest that NMDA-sensitive
mechanisms in the dentate gyrus modulate both acute and
tonic noxious sensory processing. In contrast, the hippocampal
CA1 region might modulate tonic pain behavior only.
Acknowledgment
This work was supported by a grant from the Iranian
National Science Foundation (INSF). The authors are grateful to
Dr. Yoram Shir for reviewing the manuscript and Mrs. Zahra
Deljo for her histological assistance.
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